A single-step procedure for cloning and selection of antibody-secreting hybridomas.
A procedure is described which permits high-yield direct cloning of newly established hybridomas on STO fibroblast feeders in soft agarose. Several thousand independent clones are typically obtained from each fusion (1 X 10(8) spleen cells). These are screened using a colony replica assay in which secreted antibodies diffuse through an agar overlay and bind to antigen immobilised on nitrocellulose. Bound antibodies are then detected with enzyme-labelled second antibody. The procedure is fast and efficient and permits the isolation and selection of antigen-specific clones in less than 2 weeks from fusion. It has been successfully employed for the derivation and selection of high-affinity anti-hapten antibodies. Other potential applications of the assay are in the detection of non-immobilised antigens by an indirect method using anti-globulin on nitrocellulose, in the generation of bispecific antibodies and the selection and characterisation of antibody specificities generated by the expression of antibody fragments in bacteria or yeasts.
Gherardi
E
E
Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K.
Pannell
R
R
Milstein
C
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D002999:Clone Cells; D004797:Enzyme-Linked Immunosorbent Assay; D006825:Hybridomas / Q000276:immunology*; D051379:Mice
D000911:Antibodies, Monoclonal